Wound healing requires the recruitment of specialized cells to the site of the wound for the purposes of accomplishing such tasks as blood coagulation, immune responses, inflammation and tissue repair. Platelets adhere to a wound and aid in clot formation within minutes of an injury. The phagocytic cells (leukocytes and macrophages) then debride the wound, followed by the connective tissue cells (fibroblasts and smooth muscle-like cells) which proliferate and deposit extracellular matrix. The cells involved in the wound healing process produce and/or release factors which aid in the process.
Chemotaxis is the directed migration of a cell along a gradient toward the source of a chemical. A chemoattractant is a chemical which specifically stimulates chemotaxis. It is believed that it is the sequential production of cell type-specific chemoattractants which is responsible for the ordered recruitment of phagocytes, fibroblasts and endothelial cells to the wound. For example, platelet factor 4, elastin peptides and certain synthetic N-formylmethionyl peptides attract phagocytes (neutrophils and monocytes). Fibronectin and platelet-derived growth factor summon matrix producing cells and the former also stimulates endothelial cell migration.
Several biochemical events determine a cell""s ability to respond to a chemoattractant, e.g. a growth factor. First, the chemoattractant molecules must be produced in a manner which allows them to reach the target cells desired for migration. Second, the target cells must be capable of detecting the chemoattractant, i.e. via receptors. Third, the binding of the chemoattractant to an appropriate receptor must in some way signal the target cell to move to the desired site, i.e. wound.
The proliferation of connective tissues at the wound is promoted by various types of growth factors. Competence factors (platelet derived growth factor, fibroblast growth factor and leukocyte derived growth factors) activate quiescent cells in the Go phase of the cell cycle enabling them to respond to progression factors. Progression factors, e.g. insulin, somatonedin A or C, alveolar macrophage-derived growth factor, stimulate the cells to enter the S-phase of the cell cycle.
Mitogens are agents which induce mitosis of a cell leading to cell growth or proliferation. Growth factors may be mitogenic, chemotactic, or both mitogenic and chemotactic, i.e. a mitoattractant. Even further, a growth factor may be a mitogen for one type of cells yet be a chemo- or mitoattractant for another type of cells. For example, platelet derived growth factor (PDGF) is both a chemoattractant and a mitogen for connective cells. Epidermal growth factor (EGF), transforming growth factors a and xcex2, somatonedins A and C, and insulin are growth factors which are mitogenic for fibroblast cells and yet EGF is a mitoattractant for intestinal epithelial cells. The complex nature of the functions of the various growth factors make them an interesting and important field of study.
Presently, PDGF is a growth factor which has been studied extensively. PDGF has some limitations regarding its usefulness in wound repair. First, PDGF is a dimeric glycosylated protein which is difficult and expensive to produce. Second, PDGF is a relatively large molecule which also makes it more difficult to produce recombinantly or chemically than smaller molecules.
The present invention pertains to a novel protein, Leukocyte Derived Growth Factor 2 (hereinafter LDGF2) having PDGF-like activity. LDGF2 reacts with PDGF receptors and possesses mitogenic and/or chemotactic activity for various cell types, particularly connective tissue cells. LDGF2 may be used as the active ingredient in therapeutic compositions, e.g. wound healing compositions, or even further may be used as an additive to cell culture media for the purpose of stimulating cell growth.
LDGF2 may be purified from wound fluid or may be purified as a release product of activated human monocytes. The protein has a molecular weight of about 7000 daltons determined by SDS gel electrophoresis and is about 61 amino acids in length. The relatively small size of the protein is advantageous in that it is easier to produce than its larger, glycosylated PDGF counterpart. LDGF2 also is smaller than previously discovered Leukocyte Derived Growth Factor 1 (hereinafter LDGF1).
Leukocyte Derived Growth Factor 1 (hereinafter LDGF1) was previously described in copending application Ser. No. 07/472,377 filed on Feb. 1, 1990 now abandoned. LDGF1 and LDGF2 are structurally similar but distinct proteins in that the first 49 amino acids of each protein are the same. The last 12 amino acids of LDGF2 differ significantly from the corresponding portion of LDGF1. An interesting aspect of LDGF2 is that the first 49 amino acids represent a region that is responsible for LDGF1""s activity as a connective tissue mitoattractant.
Other aspects of this invention include isolated nucleic acid, i.e. DNA, coding for LDGF2, expression vectors designed to express LDGF2, host cells transformed to express LDGF2, methods of producing LDGF2, antibodies specifically reactive with LDGF2 and therapeutic compositions and cell culture media containing LDGF2. The preferred therapeutic compositions are wound healing compositions containing a wound healing effective amount of LDGF2 and a pharmaceutically acceptable carrier.
Another important aspect of this invention pertains to the discovery that LDGF2 mRNA results from an intergenic exon exchange between the LDGF and PF4 genes and encodes two distinct polypeptides. The first being LDGF2 and the second being a fusion protein having multiple domains. The first domain of the protein is derived from a first gene (LDGF1) and the second domain is derived from a second gene (PF4) which differs from the first gene. Further, the protein exists in nature in a form that results from an intergenic exon exchange between the first and second gene that codes for a single open reading frame. The sequence of this protein is identical to that of LDGF1beginning at the methionine residue until it fuses to the sequence in exon number 2 of the PF4 gene where the sequence is identical to that of the mature PF4 peptide. This concept is particularly important for the SIG family of C-X-C proteins because it is believed that the genes responsible for these factors or proteins are localized on an area of one chromosome. Thus, it is expected that other growth factors in this family also will be the result of intergenic exon exchanges in view of the discovery that the fusion protein is the result of an intergenic exon exchange between two genes on this chromosome.